Apolipoprotein (apo) E is a major apolipoprotein associated with plasma very low density lipoprotein (VLDL), subfractions of high density lipoprotein (HDL), and with remnant particles derived from the intravascular metabolism of chylomicrons and VLDL. ApoE plays a key role in lipoprotein metabolism by acting as a ligand for hepatic receptors that mediate the rapid removal of cholesterol-laden remnant particles. The importance of apoE to human health is illustrated by the accumulation of remnant particles in patients homozygous for the apoE2 protein, an apoE allele with decreased affinity for hepatic receptors. A fraction of these individuals with an additional compromising factor develop Type III hyperlipoproteinemia and premature atherosclerosis. Unlike most plasma apolipoproteins which are synthesized only at sites of lipoprotein assembly in liver and small intestine, apoE also is synthesized at high levels in many peripheral tissues including adrenal gland, brain, skin, and ovary. The long-term goal of this research is to understand the functional basis for apoE expression in peripheral tissues. Recent studies suggest that apoE expression in adrenal cells can alter cholesterol metabolism and modulate protein kinase A (PKA) and protein kinase C (PKC) signal transduction pathways. Hypotheses derived from these findings will be tested in vivo in Aim 1 and in cell culture in Aim 2. In Aim 1 the role of apoE in adrenal cholesterol metabolism and steroidogenesis will be tested in three animal models. The first is the apoE deficient (apoEdelta) mouse in which the apoE gene has been inactivated by gene targeting. The second is a transgenic mouse (TGadrenal) in which apoE is overexpressed selectively in the adrenal cortex. Cholesteryl ester (CE) storage, CE mobilization, and steroidogenesis will be examined under basal and stimulated conditions. Immunocytochemical procedures will be used to test the hypothesis that locally-produced apoE is important for the association of HDL with the cell surface of adrenocortical cells in vivo. These studies will also determine whether cell surface apoE on adrenocortical cells is primarily derived from local apoE synthesis. The third animal model is the apoAI-deficient (apoAIdelta) mouse which exhibits an absence of CE storage in adrenal cells. ApoE regulation and the influence of local apoE expression will be tested in the apoAIdelta background. In Aim 2 primary cultures of adrenocortical cells from control, apoEdelta, and TGadrenal mice will be compared for the ability to accumulate and retain cholesterol and CE under condiitons favoring cholesterol efflux. Key parameters of the PKA and PKC signalling pathways will be tested as well as whether apoE expression alters lipoprotein utilization for steroidogenesis. Y1 cells and primary adrenal cell cultures will be used to test the influence of apoE expression on cell surface lipoprotein binding, lipoprotein uptake and processing via the endocytic pathway, and the uptake of HDL cholesterol via the nonendocytic selective uptake pathway. A Y1 cell line expressing apoE from a tightly regulated inducible promoter will be used to test the quantitative and temporal relationships between apoE expression and parameters altered by apoE. These studies are designed to evaluate the importance of localized apoE expression on tissue cholesterol metabolism and steroidogenesis in normal adrenocortical cells in vivo and in culture.